The analysis of the genome, proteome and/or methylome plays an ever-increasing role in many biological disciplines and is generally recognized as being superior to conventional methods such as, for example, the detection of metabolic products. These so-called molecular-biological analyses include, for example, medical and clinical diagnosis, forensics, the development and evaluation of drugs, foodstuff analysis, the cultivation of useful plants and environmental analysis.
Moreover, the analyses of the genome using methods such as, for example, PCR, RFLP, AFLP or sequencing, enable, for example, the detection of genetic defects or the determination of the HLA type as well as other genetic markers. Infectious pathogens such as viruses, bacteria, etc. can also be detected in this manner. The analysis of the methylome allows one to make statements about the activity of certain genes; certain methylation patterns, for example, point to the predisposition for certain diseases.
The polymerase chain reaction (PCR) is one of the most important biochemical methods for the amplification of nucleic acids. Using this technique, nucleic acids can be amplified very quickly and effectively and can then be sequenced or detected. One important field of application of PCR is clinical diagnosis.
However, in order to perform a comprehensive analysis, it is indispensable that a stable biological specimen is available so that the characteristics of the specimen are conserved. Particularly in the area of medicine, the stabilization of nucleic acids has a high priority. Specimens containing nucleic acid that are taken can frequently only be studied further after extended storage and, in some cases, transport to a laboratory. In the meantime, the nucleic acids in the specimens can change or even decompose completely. This has a massive impact on the result of tests performed later or even makes them impossible. Similarly unfavorable conditions can be found in forensics, for example, or in sampling under field conditions.
Stabilization, for example when using a stabilizing agent, should be associated with very simple and quick handling since, on the one hand, any necessary pretreatment of the specimen (e.g., washing or homogenization) prevents the immediate stabilization of the gene expression profile since, for example, RNA decomposes or is newly synthesized during the delay as a result of the pretreatment. On the other hand, any pretreatment and any additional processing step makes the use of the stabilizing agent difficult. Utilization anywhere where biological specimens are obtained, such as, for example, in the operating room, in field studies, in a food-producing business, at a crime scene, and the like is only conceivable/practical if handling is very simple and preferably does not involve the use a specific device and does not involve additional preparation of the specimen.
The purification of nucleic acids on solid phases that are based on a silica matrix is a technique used in many commercial kits. The principle of purification is based on the binding of the nucleic acids to the solid phase depending on the pH value and on the salt concentration of the buffer. Under chaotropic conditions, the network of hydrogen bridge relationships in the water is disturbed. As a result, the formation of a hydration shell around the macromolecule (DNA, RNA) is eliminated. In the absence of the chaotropic ions, a hydration shell forms again, so that the interaction between silica membrane and macromolecule is eliminated. Technically, this type of purification has been implemented e.g., in the spin-filter method and in magnetic beads technology.
The patents and patent publications referenced herein are incorporated herein by reference in their entirety.
For example, nucleic acid extraction is disclosed in U.S. Pat. No. 5,538,870. The extraction occurs basically in four steps: Cell lysis, binding of the nucleic acids to a matrix, and washing and elution of the nucleic acids. A drawback here is that the extraction is very time-consuming since, for example, numerous washing steps have to be carried out during which nucleic acid is always washed out as well. Consequently, the nucleic acid yield is reduced considerably. Furthermore, this extraction method is very difficult to automate.
Another variant of the isolation of nucleic acids using a silica matrix is disclosed, for example, in U.S. Pat. No. 6,027,945, in which magnetic silica particles are used. Molecules with a large surface are used which possess a magnetic moment when exposed to a magnetic field. Porous glasses, the surface of which has been modified with colloidal magnetite (Fe3O4) are used, among other things. These magnetic beads and a special binding buffer are added to the specimen after lysis. The nucleic acids bind to the silica matrix. Through the application of a magnetic field, the beads collect at the edge of the vessel and the impurities can be removed in several washing steps. Through the removal of the magnet and the addition of the elution buffer, the target molecules are dissolved. If a magnetic field is applied again, the elution buffer with nucleic acids can be separated from the beads. The advantage of this technique consists in the high degree to which the work sequences can be automated with low equipment costs.
U.S. Pat. No. 6,699,987 describes a kit and method for isolating nucleic acids which involves a binding to a solid phase/substrate via a lysis/binding buffer system which comprises at least one antichaotropic salt component. The antichaotropic salt component allows the nucleic acid to bind to solid phases such as glass fiber mats, glass membranes, silica carriers, ceramics, zeolites just like chaotropic materials.
A method for the stabilization of biological specimens is described, for example, in US Patent publication No. 20100255524. A biological specimen is brought in contact with a substance and stabilized by it.
Moreover, US Patent publication No. 2011092687 describes a stable lysis buffer mixture for the extraction of nucleic acids involving a storage-stable solid including controls and enzymes. The lysis buffer can be used simply for the lysis of nucleic acid-containing specimens in preparation for the purification of nucleic acids, but it can also be used for the purification itself.
Furthermore, the prior art describes means, e.g., containers, for receiving biological substances. A liquid buffer that stabilizes the nucleic acid present in a specimen over a short period of time can be introduced into these containers. In this context, liquid buffers are generally used for the stabilization via a closable cover on the funnel. Experience shows that this arrangement leads to errors during sampling, since the covers often close imprecisely and losses can occur.
Drawbacks of such systems include that they only allow storage over a short period of time (hours to days) and/or that the systems use liquid buffers. This generally results in a risk of contamination or of specimen loss.
There is a need in the art for a system with which a nucleic acid can be stored over an extended period of time, and in particular for a system that mitigates at least some of the drawbacks and shortcomings of the systems described in the prior art.